The HotStar HiFidelity Polymerase Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Efficient TA/UA cloning.

A 955 bp fragment was amplified from 100 ng of human genomic DNA using the HotStar HiFidelity Polymerase Kit and standard Taq DNA Polymerase. PCR products (4 µl each) were cloned using a commercially available TA or UA cloning kit. Blue-white screening was used to determine the cloning efficiency.

Highly sensitive and reliable PCR.

PCR was performed using HotStar HiFidelity DNA Polymerase (QIAGEN) and 4 high-fidelity PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers' recommendations, using 10 ng and 1 ng human genomic DNA. Two different high-fidelity enzymes from Supplier I were tested. A 2.3 kb fragment of the human IL9R gene was amplified in 40 PCR cycles. M: Markers.

Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.

High-fidelity enzymes generally utilize a 3'→5' exonuclease activity to remove incorrectly incorporated bases. However, this activity can also degrade primers during setup and the start of PCR, which can result in nonspecific priming, smearing, or even failure to amplify any product at all — especially when using low amounts of template.

HotStar HiFidelity PCR Buffer

Novel HotStar HiFidelity PCR Buffer contains the proprietary PCR additive, Factor SB, as well as optimized concentrations of dNTPs and MgSO4. Factor SB is a unique PCR additive that improves PCR sensitivity and reliability (see figure "Highly sensitive and reliable PCR"). Innovative Factor SB ensures that the template DNA, which is often degraded by the 3'→5' exonuclease activity of high-fidelity enzymes, remains intact.

The preoptimized formulation provides reliable amplification of specific PCR products with a very low error rate. The buffer accomplishes this by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, HotStar HiFidelity PCR Buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.

HotStar HiFidelity PCR Buffer also contains specifically optimized concentrations of dNTPs for convenient PCR setup and reliable results. The buffer also maximizes the fidelity of HotStar HiFidelity DNA Polymerase, which ensures a very low error rate. The HotStar HiFidelity Polymerase Kit is highly suitable for any high-fidelity PCR application due to the increased sensitivity, specificity, and minimal optimization requirements afforded by the HotStar HiFidelity PCR Buffer.

Q-Solution

The HotStar HiFidelity Polymerase Kit includes Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates (see figure "Amplification of difficult templates"). Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. Adding Q-Solution to the PCR does not compromise PCR fidelity.

Complete kit format

The HotStar HiFidelity Polymerase Kit includes everything required for PCR. No additional reagents or buffers need to be purchased separately.

Procedure

The HotStar HiFidelity procedure is straightforward — simply follow the step-by-step, optimized protocol included with the kit. HotStar HiFidelity DNA Polymerase has been chemically modified to temporarily inactivate not only the polymerase activity, but also the 3'–>5' exonuclease activity of the enzyme. This prevents excessive degradation of primers and template during PCR setup and the initial PCR cycles. Both polymerase and proofreading activities are easily restored by a 5-minute, 95°C incubation step, which can be readily incorporated into the PCR cycling program. This modification allows convenient room-temperature reaction setup without compromising PCR specificity or product yield.

TA/UA cloning

In contrast to all other high-fidelity DNA polymerases, PCR products generated using HotStar HiFidelity DNA Polymerase can be used directly in TA- or UA-cloning procedures (see figure "Efficient TA/UA cloning").

Product length and sensitivity

Due to the high sensitivity of the HotStar HiFidelity Polymerase Kit, it is possible to amplify fragments with very low amounts of starting material (e.g., as little as 1 ng human genomic DNA). Product lengths of up to 5 kb and 10 kb have been achieved using genomic DNA and lambda DNA respectively, thus indicating the suitability of the HotStar HiFidelity Polymerase Kit for any application, including the amplification of long DNA fragments for cloning.

Applications

HotStar HiFidelity Polymerase Kit is highly suitable for all hot-start PCR applications that require high sensitivity combined with a low error rate, such as cloning and site-directed mutagenesis.